Introduction to Liquid Chromatography

Columns System Components Applications Troubleshooting

Susan M. Steinike, M.S HPLC Marketing Department February, 2006
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Modern HPLC

Late 1970s/early 1980s

Instrumentation developed for high pressure solvent delivery: pumps, autosamplers, diode array detectors More uniform packing material produced for columns

Last 20 years

Nothing really new, but by returning to the basic theory of chromatography, even better columns are on the market: smaller particle sizes which yield faster separations, but require hardware to withstand higher pressures.
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What is Chromatography?

Separation of a mixture into individual components. The separation uses a Column (stationary phase) and Solvent (mobile phase). The components are separated from each other based on differences in affinity for the mobile or stationary phase. The goal of the separation is to have the best RESOLUTION possible between components.
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The Most Basic Explanation of Chromatography Ever

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How Do You Get Separation?

Hardware: pumps, injector, detector Column: particle diameter, column size, packing materials, and the dreaded equations Our seminar will focus on the contribution of each factor to perform separations.

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HPLC and Pre-HPLC Techniques

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Column Types

Reversed-Phase LC
Nonpolar stationary phase: C8, C18 Polar mobile phase: Water, ACN, Methanol The MOST polar compound comes out first

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Reversed Phase HPLC Columns

C-18, C-8: C-3, C-4: Phenyl: Cyano: Amino:

Rugged, general purpose, highly retentive Less retentive, used mostly for peptides & proteins Greater selectivity than alkyl-bonded Moderate retention, normal & rev. phase Weak retention, good for carbohydrates

The cyano column with a high polarity mobile phase (Water/MeOH) will act as a reversed phase column.
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Typical Column Sizes

Particle size: 5 m, 3 m, and smaller

Monodispersed means particles are the same size Very important for stable pressure and flow Smaller particles produce higher system pressure

Pore size: 100-120 A is typical Surface area: 300-350 m2/g Carbon load: 9-12% for C8, 16-20% for C18
Higher carbon load = better resolution but longer run times Lower carbon load = shorter run times, but may change selectivity vs. higher carbon load SHIMADZU

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Idealized HPLC Separation

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Capacity Factor,

The relative degree to which an analyte component is delayed as it is eluted through a given system (retentivity).
k = (Vr - V0)/V0 = (tr - t0)/t0
Where Vr = peak retention volume; V0 = column void volume tr = peak retention time; t0 = peak void time

The larger the k, the later the analyte elutes after the void.
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Mobile Phase Strength vs.

0.079
100% 100%

4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water

0.212
90% 90%

Capacity Factor for Butyl Paraben (Peak 4)

0.472
80% 80%

70% 70% 60% 60%

1.127
2.813 7.666

50% 50%

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Temperature Effect on

2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water

50C

45C

40C 35C

30C

25C

20C

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Questions About Columns?

Next HPLC System Components

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HPLC Pumps 2 Basic Types

Dual Piston
Two pistons with equal volume (10 L each) During each stroke, 10 L is delivered Best for low flow rates (< 1 mL/min) Little to NO pulsation, so its ideal for pulse sensitive detectors like RID and CDD

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Dual Piston Pump

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Sample Injection Automatic

Needle-in-the-flowpath autosampler
Sample loop and needle are a single piece of tubing Loop and needle are cleaned during the run Metering pump draws sample very precisely

Advantages: no sample loss, low carryover Disadvantages: higher cost, more delay volume for gradient
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Sample Injection to Flow Path

Sample Loading

Sample Injection Everything drawn into the needle goes to the column.

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HPLC Column Ovens

Block heater with solvent preheater

Column is housed between 2 metal plates Mobile phase is plumbed into the block for preheating

Forced air
Column is in a large chamber with air circulation Better temperature equilibration Room for column switching valves

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Why Use a Column Oven?

Retention times decrease, and higher flow rates are possible

2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water 50 C 45 C

40 C
35 C 30 C 25 C 20 C

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HPLC Detectors UV-VIS

UV-VIS

Wavelength range 190-700 nm D2 and W lamps

Most common HPLC detector for a variety of samples

Proteins and peptides Organic molecules Pharmaceuticals

Monitor 2 wavelengths at one time

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HPLC Detectors UV-VIS

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HPLC Detectors

Refractive Index

For samples with little or no UV Absorption

Alcohols, sugars, saccharides, fatty acids, polymers

Best results when RI of samples is very different from RI of mobile phase Flow cell is temperature controlled with a double insulated heating block.

REQUIRES isocratic separations REQUIRES low pulsation pumps

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HPLC Detectors RI Balance

Fill sample and reference cell with mobile phase.

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HPLC Detectors RI Analyze

Mobile phase flows through sample side only.

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HPLC Detectors RI Analyze

As the refractive index changes, the image on the photodiode is deflected or unbalanced, and the difference in current to the photodiode is measured.
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HPLC System Types

Isocratic system
Same mobile phase concentration throughout the separation Use 1 pump and pre-mix solvents Use 1 pump and a valve for 4 different solvents Use 2 pumps and vary the amount coming from each pump

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Isocratic Separation

1 pump and premixing 4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water

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Isocratic Separation

1 pump with valve and premixing

To Column
ABCD

A = 80% Methanol, 20% Water

B = 70% Methanol, 30% Water

C = 60% Methanol, 40% Water D = 50% Methanol, 50% Water
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Isocratic Separation

1 pump with mixer let the pump do the work!

To Column

ABCD

Method 1: A.CONC = 20%, B.CONC = 80%

Method 2: A.CONC = 30%, B.CONC = 70%

Method 3: A.CONC = 40%, B.CONC = 60% Method 4: A.CONC = 50%, B.CONC = 50%
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Low Pressure Gradient

1 Pump, solvents are mixed before the pump

To Column

ABCD

REQUIRES degassing
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Low Pressure Gradient

1 Pump, solvents are mixed before the pump

To Column

ABCD

REQUIRES degassing
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Questions About System Types?

Next: Troubleshooting and How to Take Care of Your Column and HPLC System
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HPLC Troubleshooting

Pressure: too much or too little Leaks: pump, autosampler, detector Reproducibility: pump, autosampler Column Care: Flushing and equilibration

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Pump Troubleshooting

No pressure, or fluctuating pressure

Pump may not be completely full of liquid check solvent inlet line Air in check valve always degas mobile phase! Stuck check valve the pump may have been idle for too long and solvent has dried inside the check valve. Poor quality solvent: may contain resins that coat the ball inside the check valve, and that film wont let the ball seat properly

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Pump Troubleshooting

High Pressure

Outlet frit may be blocked with particles from mobile phase or seal material

Leaks

Damage to seal and/or plunger due to several factors

Misaligned plunger Solvent incompatibility with seal material Salt crystal buildup from buffers use a rinse kit!

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Pump Troubleshooting

Retention Time Reproducibility

For a dual piston pump, only one side may be filled with liquid check solvent inlet lines Temperature change (may not be the pumps fault)

A 1o shift in temperature can result in a 1-2% shift in retention time Avoid drafty locations in the lab Use a column oven when possible

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Autosampler Troubleshooting

High Pressure

Particulates from mobile phase, sample, pump may be trapped in the inlet tubing or valve

Filter mobile phase AND sample when possible

Leaks

Fittings may be loose on the valve

Tighten fittings properly and dont exceed the pressure limit of the autosampler
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Autosampler Troubleshooting

Area % Reproducibility
Always degas rinse phase, and use some volume of liquid for rinsing to keep all flow paths in the valves full of liquid Make sure the needle stroke is deep enough to draw sample from the vial Check for leaks on the valve fittings, and the connection to the column inlet

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Detector Troubleshooting

Spiky Baseline

Air bubble in flow cell degas mobile phase!

Put some restriction on the cell outlet, but not too much! Tubing with 0.005 i.d. is fine.

Leaks

Cracked flow cell

Dont exceed the pressure limit of the cell Use the proper fittings and tighten appropriately
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Poor tubing connections

Column Care

Follow MFRs recommendations for solvent compatibility, flow rate, and pressure limits Filter samples when possible

Particulates will build up on the inlet frit over time Connect the outlet to waste, NOT inline with the detector to prevent further contamination

Use care when reversing column flow

Store columns in recommended solvents

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Troubleshooting Summary

Throw away bad parts and columns. Leaks do not fix themselves. If it doesnt pass, you must degas.

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